CN106520964A - Double-recognition quantitative detection method for microRNA - Google Patents
Double-recognition quantitative detection method for microRNA Download PDFInfo
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Abstract
The invention discloses a method for achieving micronRNA single-step, phase homogenizing and high-sensitivity detection by combining different double recognition processes of hybridization and ligation based on an upconversion luminescence resonance energy transfer (UC-LRET) technology. The method is mainly applied to microRNA expression profiles analysis, microRNA clinical diagnosis, microRNA study detection and microRNA related pharmaceutical studying and screening. The method has the following beneficial effects that the number of operation steps is small, and the method belongs to one-step method detection; the stability is good, long-time storage can be achieved, and the detecting performance is not reduced; and stimulating occurs in a near infrared region (980 nm), so that generation of background fluorescence is avoided. Thus, the method has very good application prospects.
Description
Technical field
The invention belongs to biotechnology and clinical diagnose field, are related to the quantitative of a kind of couple of recognition detection microRNA
Detection method.Based on up-conversion fluorescence resonance energy transfer (UC-LRET) technology and with reference to different hybridization
(hybridization) (ligation) double recognition detections, are connected, so as to realize to microRNA single steps, homogeneous, highly sensitive inspection
Survey.It is mainly used in microRNA expression pattern analysis, microRNA clinical diagnosises, microRNA researchs detection and microRNA phases
Close drug research and screening.
Background technology
MicroRNA (hereinafter referred " miRNA ") is the non-coding RNA that a class length is about 20-24 nucleotide (nt),
It can regulate and control the human gene more than half.[referring to (a) D.P.Bartel, Cell, 2009,136,215-233. (b)
S.L.Ameres, M.D.Horwich, J.H.Hung, J.Xu, M.Ghildiyal, Z.Weng and P.D.Zamore,
Science, 2010,328,1534-1539. (c) Y.Chen, D.Y.Gao and L.Huang, Adv.Drug Delivery
Rev.2015,81,128. (d) R.Devulapally, N.M.Sekar, T.V.Sekar, K.Foygel, T.F.Massoud,
J.R.K.Willmann and R.Paulmurugan, ACS Nano, 2015,9,2290.] identical miRNA is in different groups
Knit, there is similar adjusting function, with conservative between organ and different cells.But different tissues organ, different cells
And cytocerastic different phase, the express spectra of its miRNA simultaneously differs, and this cell and " space-time " specificity make which can be with
As the specific molecular markers of some histoorgans, different cells and cell development different phase.In recent years, miRNA is more
It is considered diagnosis and treats the mark of new generation and target of some diseases.[referring to (a) R.Ma, T.Jiang and
X.Kang, J.Exp.Clin.Cancer Res.2012,31,38. (b) Y.Cao, R.A.DePinho, M.Ernst and
K.Vousden, Nat.Rev.Cancer, 2011,11,749.] miRNA has become study hotspot in recent years, however, due to
Very low concentration that miRNA has, it is easy to degraded, short chain (18-24nt) and very strong sequence similarity these features
So that sensitive, specific detection miRNA becomes extremely difficult.At present, micro-array chip " hybridization " is (hybridization) and base
In quantitative real-time polymerase chain reaction (PCR) technology be the most common technology of two kinds of detection by quantitative miRNA.[referring to A.Git,
H.Dvinge, M.Salmon-Divon, M.Osborne, C.Kutter, J.Hadfield, P.Bertone and C.Caldas,
RNA, 2010,16,991.].However, the miRNA melting temperature diversityes caused by sequence polymorphism cause hybridization
Based on technology be difficult to apply to large-scale expression analysis, and the technology based on PCR needs miRNA labellings and complementation
Multiple programs such as DNA step of converting so that detection miRNA is time-consuming, effort.[referring to (a) Z.Gao, H.Deng, W.Shen and
Y.Ren, Anal.Chem.2013,85,1624-1630. (b) Y.Ren, H.Deng, W.Shen and Z.Gao,
Anal.Chem.2013,85,4784-4789.] therefore, develop quick, simple and sensitive biological detection miRNA technology and giving birth to
Thing is studied significant with clinical diagnosises.
The content of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the invention provides a kind of be based on up-conversion fluorescence resonance energy transfer
(UC-LRET) method of the highly sensitive detection miRNA of technology, it can provide under biotic environment " without background " and homogeneously detect,
And the special of detection is caused by different hybridization " hybridization " and fusion " ligation steps " double identification steps
Attribute is greatly improved.
The present invention is made up of signal generation unit and recognition unit, upper conversion nano granule (NaYF4：Er3+, Yb3+) mark
Donor of the oligonucleotide of note as fluorescence, and the oligonucleotide that dyestuff Cy3 is coupled is used as the acceptor of fluorescence, donor-acceptance
Body has collectively constituted a pair of up-conversion fluorescence resonance energy transfer oligonucleotide pair, used as signal generation unit；Recognition unit bag
Include two sections of sequences to target miRNA with specific recognition.In the case where near infrared light is excited (980nm), up-conversion fluorescence is produced
Resonance energy transfer signal, can realize the high sensitivity to miRNA, high selectivity detection.
Using technical scheme：
A kind of upper conversion nano granule (NaYF for detection by quantitative miRNA4：Er3+, Yb3+) labelling oligonucleotide
(LRET-B), wherein described oligonucleotide sequence from 5 ' end start be and the specific sequence of recognition unit complementary pairing, control
The sequence of oligonucleotide length processed and 3 ' terminal modified C7 amino sequences compositions.
A kind of oligonucleotide (LRET-A) of the dye molecule Cy3 labellings for detection by quantitative miRNA, wherein described widow
Nucleotide sequence has the special of the sequence and recognition unit complementary pairing of Cy3 control oligonucleotide lengths from 5 ' end beginning labels
Property sequence and 3 ' end C6 modification.
A kind of recognition unit oligonucleotide (RD-REG-D) for detection by quantitative miRNA, wherein described oligonucleotide
Sequence is from PO4The specific sequence and oligonucleotide (LRET-B) that 5 ' ends of modification start with target miRNA complementary pairing is complementary
The specific sequence of pairing and 3 ' end compositions.
A kind of recognition unit oligonucleotide (RA-REG-A) for detection by quantitative miRNA, wherein described oligonucleotide
Sequence starts the specific sequence with oligonucleotide (LRET-A) complementary pairing and the spy of target miRNA complementary pairing from 5 ' ends
Different in nature sequence and be modified with-OH groups 3 ' end composition.
A kind of method of the highly sensitive detection miRNA based on up-conversion fluorescence resonance energy transfer (UC-LRET) technology, presses
Following steps are carried out：
(1), upper conversion nano granule (NaYF4：Er3+, Yb3+) labeled oligonucleotide (LRET-B)：1～3ml concentration is 0.2
Add 1- (3- dimethylamino-propyls) -3- ethyls carbon two in～2mg/ml carboxylated upper conversion nano granule UCNPs solution sub-
The N-hydroxy-succinamide (NHS) of amine hydrochlorate (EDC) 2～6mg and 1～4mg, vibrates at room temperature 60 minutes, then will
During oligonucleotide (LRET-B) solution of 0.5～2 μM of 50～150 μ L adds above-mentioned solution, reaction 12～24 is little at room temperature
When.After completion of the reaction, ultrafiltration centrifugation, product are stored in containing in 0.1% bovine serum albumin BSA buffer (pH7.4).
(2), the detection by quantitative based on double identification microRNA：The upper conversion nano granule (NaYF of 5～15nM4：Er3+, Yb3+)
The oligonucleotide (LRET-B) of labelling, the oligonucleotide (LRET-A) of 5～15nM dye molecule Cy3 labellings, two kinds of 5～15nM are double
Recognition unit oligonucleotide (RA-REG-A), (RD-REG-D), 0.02～0.12CEU RNA ligases and certain density mesh
Mark miRNA is blended in 100 μ L miRNA buffer, and miRNA buffer consists of (25mM HEPES buffer solutions, 0.4mM
ATP, 50mM NaCl, 2mM MgCl2, 100 μ g/ml single stranded salmon sperm DNA, pH7.4+0.1%
BSA).Biased sample is incubated 60～120min at 20～40 DEG C, finally carries out up-conversion fluorescence quantitative analyses.Exciting light is used
The LASER Light Source of 980nm is excited, and power is adjusted between 0～3W.
It is compared with existing " microarray hybridization method " and " quantitative pcr amplification method ", provided by the present invention based on upper conversion
The method of the highly sensitive detection miRNA of Fluorescence Resonance Energy transfer (UC-LRET) technology has advantages below：
(1) operating procedure is few, belongs to " one-step method " detection.And " microarray hybridization method " and " quantitative pcr amplification method " needs
Hybridization, amplification and its dyeing and dyeing, analytical procedure are more, easily malfunction in the process.
(2) good stability, can be preserved for a long time, and detection performance does not decline.
(3) due to exciting near infrared region (980nm), it is to avoid the generation of background fluorescence.
During invention is completed, select miRNA-21, miRNA-125a, miRNA-125b as detection object,
Detect that the property indices of the method for miRNAs carried out based on up-conversion fluorescence resonance energy transfer (UC-LRET) to described
Test repeatedly.As a result show, for the range of linearity of miRNAs detections is 200pM～1.4nM, seven times measurement reproducibility is
3.9%, in tumor cell Hela extracting solution in the detection of miRNA-21, the response rate is 93-105%.
Description of the drawings
Fig. 1 is shown based on the highly sensitive detection miRNAs of up-conversion fluorescence resonance energy transfer (UC-LRET) technology for detection
It is intended to.
Fig. 2 is upper conversion nano granule (NaYF4：Er3+, Yb3+) transmission electron microscope picture A and high-resolution-ration transmission electric-lens figure B；
Fig. 3 (A) is the fluorescence spectra and (B) linear relationship curve chart for measuring miRNA-21.
Specific embodiment
Form, is described in further detail again to the above of the present invention by the following examples, but should not be by this
The scope for being interpreted as above-mentioned theme of the invention is only limitted to Examples below, and all technologies realized based on the above of the present invention are equal
Belong to the scope of the present invention.
Below in conjunction with the accompanying drawings, the present invention is described in further detail by specific embodiment.
Embodiment 1：Upper conversion nano granule (NaYF4：Er3+, Yb3+) labeled oligonucleotide (LRET-B)
Experiment material：Bovine serum albumin, lot number are 140318, Shanghai Aladdin reagent company limited；1- ethyls-(3-
Dimethylaminopropyl) carbodiimide hydrochloride (EDC), lot number 090M14531V, Shanghai Aladdin reagent company limited；Succinum
Acid imide (NHS), lot number MKBG7914V, Shanghai Aladdin reagent company limited；Ethanol, lot number is：14021710247, it is purchased from
Nanjing Chemistry Reagent Co., Ltd..Oligonucleotide sequence (3 ' C7- of such as 5 '-TTGTGTTCCGATAGGCT-AAAAAAAA
NH2) start to be to repair with the specific sequence of recognition unit complementary pairing, the sequence of control oligonucleotide length and 3 ' ends from 5 ' ends
Decorations C7 amino sequence compositions, wherein TTGTGTTCCGATAGGCT is the specific sequence with recognition unit complementary pairing, with detection
MiRNA it is different and change.Oligonucleotide sequence is：
UC-LRET-B：5’ -TTGTGTTCCGATAGGCT-AAAAAAAA 3’ C7-NH2；
UC-LRET-A：Cy3-5’ AAAAAAAA-CGATCAGTCAGGCAA-3’ C6；
the adaptor oligos(for miRNA-21)：
3’ TCCCCAACACAAGGCTATCCGA-ATCGAATAGTCT-5’ -PO4
3’ OH-GACTACAACT-GCTAGTCAGTCCGTTTCGCC5’
(for miRNA-125a)：
3’ TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5’ -PO4
3’ OH-AAATTGGACACT-GCTAGTCAGTCCGTTTCGCC5’
(for miRNA-125b)：
3’ TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5’ -PO4
3’ OH-ATTGAACACT-GCTAGTCAGTCCGTTTCGCC5’
miRNA-125a：5’-UCCCUGAGACCCUUUAACCUGUGA-3’
miRNA-125b：5’-UCCCUGAGACCCUAACUUGUGA-3’
miRNA-21：5’-UAGCUUAUCAGACUGAUGUUGA-3
Experimental apparatus and equipment：KQ-500DE ultrasonic cleaners, DHG-9143BS electric drying oven with forced convections, AY120 are electric
Sub- analytical balance, TGL-16C centrifuges.
Experimentation：1ml concentration is the 1- of addition 2mg in the carboxylated upper conversion nano granule UCNPs solution of 1mg/ml
The N-hydroxy-succinamide (NHS) of (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) and 1mg, in room temperature
Then 1 μM of 100 μ L of oligonucleotide (LRET-B) solution added in above-mentioned solution, at room temperature instead by lower vibration 60 minutes
Answer 18 hours.Oligonucleotide sequence (3 ' C7-NH of such as 5 '-TTGTGTTCCGATAGGCT-AAAAAAAA2) from 5 ' end start for
Specific sequence, the sequence of control oligonucleotide length and 3 ' terminal modified C7 amino sequences groups with recognition unit complementary pairing
Into wherein TTGTGTTCCGATAGGCT is the specific sequence with recognition unit complementary pairing.After completion of the reaction, ultrafiltration centrifugation,
Product is stored in containing in 0.1% bovine serum albumin BSA buffer (pH7.4).Upper conversion nano granule after labelling
(NaYF4：Er3+, Yb3+) carry out transmission electron microscope and high-resolution-ration transmission electric-lens detection.As shown in Figure 2.
Embodiment 2：Detection by quantitative miRNA-21 based on double identification microRNAs
Experiment material：Ethanol, lot number is：14021710247, purchased from Nanjing Chemistry Reagent Co., Ltd..HEPES is buffered
Liquid, lot number is：E607018, purchased from Shanghai Sheng Gong biological reagents company limited；5 ' adenosine disodium triphosphate, three water (ATP), batch
Number it is：A600020, purchased from Shanghai Sheng Gong biological reagents company limited；Single stranded salmon sperm DNA, batch
Number it is：C640031, purchased from Shanghai Sheng Gong biological reagents company limited；RNA ligase, lot number is：B101801, gives birth to purchased from Shanghai
Work biological reagent company limited.
Oligonucleotide sequence is：
UC-LRET-B：5’-TTGTGTTCCGATAGGCT-AAAAAAAA 3’ C7-NH2；
UC-LRET-A：Cy3-5’ AAAAAAAA-CGATCAGTCAGGCAA-3’ C6；
the adaptor oligos(for miRNA-21)：
(RD-REG-A)：3’ TCCCCAACACAAGGCTATCCGA-ATCGAATAGTCT-5’ -PO4
(RD-REG-D)：3’ OH-GACTACAACT-GCTAGTCAGTCCGTTTCGCC5’
miRNA-21：5’-UAGCUUAUCAGACUGAUGUUGA-3
Experimental apparatus and equipment：KQ-500DE ultrasonic cleaners, DHG-9143BS electric drying oven with forced convections, AY120 are electric
Sub- analytical balance, TGL-16C centrifuges.
Experimentation：The upper conversion nano granule (NaYF of 5nM4：Er3+, Yb3+) labelling oligonucleotide (LRET-B), 5nM dye
The oligonucleotide (LRET-A) of material molecule Cy3 labellings, two kinds of 5nM double recognition unit oligonucleotide (RA-REG-A), (RD-REG-
D), 0.10CEU RNA ligases and certain density target miRNA-21 are blended in 100 μ L miRNA buffer, miRNA
Buffer consists of (25mM HEPES buffer solutions, 0.4mM ATP, 50mM NaCl, 2mM MgCl2, 100 μ g/ml single
Stranded salmon sperm DNA, pH7.4+0.1%BSA).Biased sample is incubated 90min at 25 DEG C, finally carries out
Conversion fluorescence quantitative analyses.Exciting light is excited with the LASER Light Source of 980nm, and power is adjusted between 0～3W.(what is measured is upper
Conversion fluorescence spectrogram and calibration graph are as shown in Figure 3)
Embodiment 3：Detection by quantitative miRNA-125a based on double identification microRNAs
Experiment material：Ethanol, lot number is：14021710247, purchased from Nanjing Chemistry Reagent Co., Ltd..HEPES is buffered
Liquid, lot number is：E607018, purchased from Shanghai Sheng Gong biological reagents company limited；5 '-adenosine disodium triphosphate, three water (ATP),
Lot number is：A600020, purchased from Shanghai Sheng Gong biological reagents company limited；Single stranded salmon sperm DNA,
Lot number is：C640031, purchased from Shanghai Sheng Gong biological reagents company limited；RNA ligase, lot number is：B101801, purchased from Shanghai
Sheng Gong biological reagents company limited；
Oligonucleotide sequence is：
UC-LRET-B：5’-TTGTGTTCCGATAGGCT-AAAAAAAA 3’ C7-NH2；
UC-LRET-A：Cy3-5’ AAAAAAAA-CGATCAGTCAGGCAA-3’ C6；
the adaptor oligos(for miRNA-125a)：
(RD-REG-A)：3’ TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5’ -PO4
(RD-REG-D)：3’ OH-AAATTGGACACT-GCTAGTCAGTCCGTTTCGCC 5’
miRNA-125a：5’-UCCCUGAGACCCUUUAACCUGUGA-3
Experimental apparatus and equipment：KQ-500DE ultrasonic cleaners, DHG-9143BS electric drying oven with forced convections, AY120 are electric
Sub- analytical balance, TGL-16C centrifuges.
Experimentation：The upper conversion nano granule (NaYF of 5nM4：Er3+, Yb3+) labelling oligonucleotide (LRET-B), 5nM dye
The oligonucleotide (LRET-A) of material molecule Cy3 labellings, two kinds of 5nM double recognition unit oligonucleotide (RA-REG-A), (RD-REG-
D), 0.10CEU RNA ligases and certain density target miRNA-125a are blended in 100 μ L miRNA buffer,
MiRNA buffer consists of (25mM HEPES buffer solutions, 0.4mM ATP, 50mM NaCl, 2mM MgCl2, 100 μ g/ml
Single stranded salmon sperm DNA, pH7.4+0.1%BSA).Biased sample is incubated 90min at 25 DEG C, finally
Carry out up-conversion fluorescence quantitative analyses.Exciting light is excited with the LASER Light Source of 980nm, and power is adjusted between 0～3W.
Embodiment 4：Detection by quantitative miRNA-125b based on double identification microRNAs
Experiment material：Ethanol, lot number is：14021710247, purchased from Nanjing Chemistry Reagent Co., Ltd..HEPES is buffered
Liquid, lot number is：E607018, purchased from Shanghai Sheng Gong biological reagents company limited；5 '-adenosine disodium triphosphate, three water (ATP),
Lot number is：A600020, purchased from Shanghai Sheng Gong biological reagents company limited；Single stranded salmon sperm DNA,
Lot number is：C640031, purchased from Shanghai Sheng Gong biological reagents company limited；RNA ligase, lot number is：B101801, purchased from Shanghai
Sheng Gong biological reagents company limited；
Oligonucleotide sequence is：
UC-LRET-B：5’-TTGTGTTCCGATAGGCT-AAAAAAAA 3’ C7-NH2；
UC-LRET-A：Cy3-5’ AAAAAAAA-CGATCAGTCAGGCAA-3’ C6；
the adaptor oligos(for miRNA-125b)：
(RD-REG-A)：3’ TCCCCAACACAAGGCTATCCGA-AGGGACTCTGGG-5’ -PO4
(RD-REG-D)：3’ OH-ATTGAACACT-GCTAGTCAGTCCGTTTCGCC5’
miRNA-125b：5’-UCCCUGAGACCCUAACUUGUGA-3’
Experimental apparatus and equipment：KQ-500DE ultrasonic cleaners, DHG-9143BS electric drying oven with forced convections, AY120 are electric
Sub- analytical balance, TGL-16C centrifuges.
Experimentation：The upper conversion nano granule (NaYF of 8nM4：Er3+, Yb3+) labelling oligonucleotide (LRET-B), 8nM dye
The oligonucleotide (LRET-A) of material molecule Cy3 labellings, two kinds of 8nM double recognition unit oligonucleotide (RA-REG-A), (RD-REG-
D), 0.10CEU RNA ligases and certain density target miRNA-125b are blended in 100 μ L miRNA buffer,
MiRNA buffer consists of (25mM HEPES buffer solutions, 0.4mM ATP, 50mM NaCl, 2mM MgCl2,100 μ g/ml
Single stranded salmon sperm DNA, pH7.4+0.1%BSA).Biased sample is incubated 90min at 30 DEG C, finally
Carry out up-conversion fluorescence quantitative analyses.Exciting light is excited with the LASER Light Source of 980nm, and power is adjusted between 0～3W.
The quantitative relationship figure for obtaining is as shown in Figure 3.As a result show, for miRNAs detection the range of linearity be 0.2pM～
1.4nM, 7 times measurement reproducibility is 3.9%.
Above example is merely to illustrate the preferred embodiment of the present invention, but the present invention is not limited to above-mentioned embodiment party
Formula, in the ken that the field those of ordinary skill possesses, that what is made within the spirit and principle of wood invention is any
Modification, equivalent substitute and improvement etc., which all should be within the scope of the technical scheme that the present invention is claimed.
Claims (2)
1. the quantitative detecting method of a kind of couple of recognition detection microRNA, it is characterised in that carry out according to the following steps：
(1), upper conversion nano granule (NaYF4：Er3+, Yb3+) labeled oligonucleotide (LRET-B)：1～3ml concentration be 0.2～
2mg/ml carboxylated upper conversion nano granule (NaYF4：Er3+, Yb3+) add 1- (3- dimethylamino-propyls) -3- second in solution
The N-hydroxy-succinamide (NHS) of base carbodiimide hydrochloride (EDC) 2～6mg and 1～4mg, vibrates 60 points at room temperature
Clock, then adds 0.5～2 μM of 50～150 μ L of oligonucleotide (LRET-B) solution in above-mentioned solution, reacts at room temperature
12～24 hours.After completion of the reaction, ultrafiltration centrifugation, product are stored in containing in 0.1% bovine serum albumin BSA buffer
(pH7.4)。
(2), the detection by quantitative based on double identification microRNA：The upper conversion nano granule (NaYF of 5～15nM4：Er3+, Yb3+) labelling
Oligonucleotide (LRET-B), the oligonucleotide (LRET-A) of 5～15nM dye molecule Cy3 labellings, the double identifications of two kinds of 5～15nM
Unit oligonucleotide (RA-REG-A), (RD-REG-D), 0.02～0.12CEU RNA ligases and certain density target
MiRNA is blended in 100 μ L miRNA buffer, miRNA buffer consist of (25mM HEPES buffer solutions, 0.4mM ATP,
50mM NaCl, 2mM MgCl2, 100 μ g/ml single stranded salmon sperm DNA, pH7.4+0.1%
BSA).Biased sample is incubated 60～120min at 20～40 DEG C, finally carries out up-conversion fluorescence quantitative analyses.
2. the quantitative detecting method of a kind of couple of recognition detection microRNA according to claim 1, it is characterised in that：
(1) with upper conversion nano granule (NaYF4：Er3+, Yb3+) labelling oligonucleotide (LRET-B), its sequence be 5 '-
TTGTGTTCCGATAGGCT-AAAAAAAA3’C7-NH2, wherein 3 ' end C7-NH2Modification, the sequence such as SEQ ID NO：1 institute
Show.
(2) oligonucleotide (LRET-A) of dye molecule Cy3 labellings, its sequence are Cy3-5 ' AAAAAAAA-
CGATCAGTCAGGCAA-3 ' C6, wherein 5 ' end Cy3 modifications, 3 ' end C6 modifications, the sequence such as SEQ ID NO：Shown in 2.
(3) double recognition unit oligonucleotide (RA-REG-A), its sequence is：
To detect miRNA-21：5’PO4- TCTGATAAGCTA-AGCCTATCGGAACACAACCCCT-3 ', wherein 5 ' end PO4
Modification, the sequence such as SEQ ID NO：Shown in 3.
To detect miRNA-125a：5’PO4- GGGTCTCAGGGA-AGCCTATCGGAACACAACCCCT-3 ', wherein 5 ' ends
PO4Modification, the sequence such as SEQ ID NO：Shown in 4.
To detect miRNA-125b：5’PO4-GGGTCTCAGGGA-AGCCTATCGGAACACAACCCCT-3’；Wherein 5 ' ends
PO4Modification, the sequence such as SEQ ID NO：Shown in 5.
(4) double recognition unit oligonucleotide (RA-REG-D), its sequence is：
To detect miRNA-21：5’-CCGCTTTGCCTGACTGATCG-TCAACATCAG-OH3’；Wherein 3 ' end OH modifications,
The sequence such as SEQ ID NO：Shown in 6.
To detect miRNA-125a：5’-CCGCTTTGCCTGACTGATCG-TCACAGGTTAAA-OH3’；Wherein 3 ' end OH are repaiied
Decorations, the sequence such as SEQ ID NO：Shown in 7.
To detect miRNA-125b：5’-CCGCTTTGCCTGACTGATCG-TCACAAGTTA-OH3’；Wherein 3 ' end OH are repaiied
Decorations, the sequence such as SEQ ID NO：Shown in 8.
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Cited By (3)
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| CN108037100A (en) * | 2017-11-16 | 2018-05-15 | 江南大学 | A kind of method that two kinds of miRNA are detected while the effect based on FRET |
| CN110452961A (en) * | 2018-12-03 | 2019-11-15 | 天津大学 | It is a kind of for detecting the preparation method and application of the hydrogel of miRNA |
| CN112997965A (en) * | 2021-03-08 | 2021-06-22 | 国家卫生健康委科学技术研究所 | CRISPR/Cas9 technology-based miRNA-125a knockout mouse model and construction method |
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| CN110452961A (en) * | 2018-12-03 | 2019-11-15 | 天津大学 | It is a kind of for detecting the preparation method and application of the hydrogel of miRNA |
| CN112997965A (en) * | 2021-03-08 | 2021-06-22 | 国家卫生健康委科学技术研究所 | CRISPR/Cas9 technology-based miRNA-125a knockout mouse model and construction method |
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